CN106879462B - A kind of Ranalisma rostratum micro-propagation method - Google Patents
A kind of Ranalisma rostratum micro-propagation method Download PDFInfo
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- 241001408209 Ranalisma rostratum Species 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 19
- 210000000056 organ Anatomy 0.000 claims abstract description 7
- 238000002054 transplantation Methods 0.000 claims abstract description 5
- 238000004140 cleaning Methods 0.000 claims abstract description 3
- 239000001963 growth medium Substances 0.000 claims description 39
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims description 9
- 229920001817 Agar Polymers 0.000 claims description 8
- 229930186147 Cephalosporin Natural products 0.000 claims description 8
- 239000008272 agar Substances 0.000 claims description 8
- 229940124587 cephalosporin Drugs 0.000 claims description 8
- 150000001780 cephalosporins Chemical class 0.000 claims description 8
- HZZVJAQRINQKSD-PBFISZAISA-N clavulanic acid Chemical compound OC(=O)[C@H]1C(=C/CO)/O[C@@H]2CC(=O)N21 HZZVJAQRINQKSD-PBFISZAISA-N 0.000 claims description 8
- OHKOGUYZJXTSFX-KZFFXBSXSA-N ticarcillin Chemical compound C=1([C@@H](C(O)=O)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)C=CSC=1 OHKOGUYZJXTSFX-KZFFXBSXSA-N 0.000 claims description 8
- 229960004659 ticarcillin Drugs 0.000 claims description 8
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 6
- 239000005556 hormone Substances 0.000 claims description 6
- 229940088597 hormone Drugs 0.000 claims description 6
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 5
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 5
- 230000001954 sterilising effect Effects 0.000 claims description 5
- 239000000126 substance Substances 0.000 claims description 5
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 4
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 3
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 claims description 3
- 239000004471 Glycine Substances 0.000 claims description 3
- 239000007836 KH2PO4 Substances 0.000 claims description 3
- 229910004619 Na2MoO4 Inorganic materials 0.000 claims description 3
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims description 3
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 3
- 238000009395 breeding Methods 0.000 claims description 3
- 230000001488 breeding effect Effects 0.000 claims description 3
- 239000001110 calcium chloride Substances 0.000 claims description 3
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 3
- 229910052927 chalcanthite Inorganic materials 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims description 3
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 3
- 229910052603 melanterite Inorganic materials 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 229960003512 nicotinic acid Drugs 0.000 claims description 3
- 235000001968 nicotinic acid Nutrition 0.000 claims description 3
- 239000011664 nicotinic acid Substances 0.000 claims description 3
- 239000011684 sodium molybdate Substances 0.000 claims description 3
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 3
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 claims description 3
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 3
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 3
- 239000011686 zinc sulphate Substances 0.000 claims description 3
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims description 2
- 229930006000 Sucrose Natural products 0.000 claims description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 239000005720 sucrose Substances 0.000 claims description 2
- 235000019190 thiamine hydrochloride Nutrition 0.000 claims description 2
- 239000011747 thiamine hydrochloride Substances 0.000 claims description 2
- 229960000344 thiamine hydrochloride Drugs 0.000 claims description 2
- 229960005486 vaccine Drugs 0.000 claims 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims 1
- 230000003796 beauty Effects 0.000 claims 1
- 230000004083 survival effect Effects 0.000 abstract description 4
- 238000010413 gardening Methods 0.000 abstract description 2
- 241000196324 Embryophyta Species 0.000 description 22
- JTEDVYBZBROSJT-UHFFFAOYSA-N indole-3-butyric acid Chemical compound C1=CC=C2C(CCCC(=O)O)=CNC2=C1 JTEDVYBZBROSJT-UHFFFAOYSA-N 0.000 description 16
- 238000007792 addition Methods 0.000 description 9
- 230000000694 effects Effects 0.000 description 8
- 230000006698 induction Effects 0.000 description 8
- 238000010586 diagram Methods 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 230000003115 biocidal effect Effects 0.000 description 4
- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 206010020649 Hyperkeratosis Diseases 0.000 description 3
- 238000005286 illumination Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 2
- 238000000692 Student's t-test Methods 0.000 description 2
- NNBFNNNWANBMTI-UHFFFAOYSA-M brilliant green Chemical compound OS([O-])(=O)=O.C1=CC(N(CC)CC)=CC=C1C(C=1C=CC=CC=1)=C1C=CC(=[N+](CC)CC)C=C1 NNBFNNNWANBMTI-UHFFFAOYSA-M 0.000 description 2
- 230000032823 cell division Effects 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 238000004851 dishwashing Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000000763 evoking effect Effects 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 229960000367 inositol Drugs 0.000 description 2
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 2
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 2
- RADKZDMFGJYCBB-UHFFFAOYSA-N pyridoxal hydrochloride Natural products CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 description 2
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 229960003495 thiamine Drugs 0.000 description 2
- 238000012549 training Methods 0.000 description 2
- 235000019158 vitamin B6 Nutrition 0.000 description 2
- 239000011726 vitamin B6 Substances 0.000 description 2
- 229940011671 vitamin b6 Drugs 0.000 description 2
- 239000012224 working solution Substances 0.000 description 2
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 206010023126 Jaundice Diseases 0.000 description 1
- FAIXYKHYOGVFKA-UHFFFAOYSA-N Kinetin Natural products N=1C=NC=2N=CNC=2C=1N(C)C1=CC=CO1 FAIXYKHYOGVFKA-UHFFFAOYSA-N 0.000 description 1
- 241000544002 Ranalisma Species 0.000 description 1
- JZRWCGZRTZMZEH-UHFFFAOYSA-N Thiamine Natural products CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 description 1
- 229930003451 Vitamin B1 Natural products 0.000 description 1
- 150000005018 aminopurines Chemical class 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- QANMHLXAZMSUEX-UHFFFAOYSA-N kinetin Chemical compound N=1C=NC=2N=CNC=2C=1NCC1=CC=CO1 QANMHLXAZMSUEX-UHFFFAOYSA-N 0.000 description 1
- 229960001669 kinetin Drugs 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000006193 liquid solution Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 229960002523 mercuric chloride Drugs 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 description 1
- 230000003020 moisturizing effect Effects 0.000 description 1
- 239000003415 peat Substances 0.000 description 1
- 239000003375 plant hormone Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 235000019157 thiamine Nutrition 0.000 description 1
- 239000011721 thiamine Substances 0.000 description 1
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 description 1
- UZKQTCBAMSWPJD-UQCOIBPSSA-N trans-Zeatin Natural products OCC(/C)=C\CNC1=NC=NC2=C1N=CN2 UZKQTCBAMSWPJD-UQCOIBPSSA-N 0.000 description 1
- UZKQTCBAMSWPJD-FARCUNLSSA-N trans-zeatin Chemical compound OCC(/C)=C/CNC1=NC=NC2=C1N=CN2 UZKQTCBAMSWPJD-FARCUNLSSA-N 0.000 description 1
- 239000010455 vermiculite Substances 0.000 description 1
- 229910052902 vermiculite Inorganic materials 0.000 description 1
- 235000019354 vermiculite Nutrition 0.000 description 1
- 235000010374 vitamin B1 Nutrition 0.000 description 1
- 239000011691 vitamin B1 Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000004383 yellowing Methods 0.000 description 1
- 229940023877 zeatin Drugs 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The present invention relates to a kind of Ranalisma rostratum micro-propagation methods, belong to gardening technical field of forestry.It is sterilized the Ranalisma rostratum after cleaning to obtain aseptic seedling as explant, micro- numerous organ is chosen in aseptic seedling and carries out culture of rootage, obtained tissue culture transplantation of seedlings.Invention is applied to the culture of Ranalisma rostratum, has many advantages, such as to cultivate fast, high survival rate.
Description
Technical field
The present invention relates to a kind of Ranalisma rostratum micro-propagation methods, belong to gardening technical field of forestry.
Background technique
Ranalisma rostratum (Ranalisma rostratum) it is a kind of aquatic herbaceous plant in imminent danger, it is subordinate to Alismataceae
Ranalisma originates in China Zhejiang, Jiangxi, Hunan and Vietnam and Malaysia, domestic only to have found at present in Jiangxi, Hunan
Scattered is small gregarious, and gregarious number and individual are still reducing, and illustrate that this plant has been in serious Critical Condition.Mesh
Before, the organizational project nursery about Ranalisma rostratum is rarely reported.And how to expand its population quantity, and carry out to the plant
Protection, is urgently to solve the problems, such as.
Based on this, the application is made.
Summary of the invention
For the status of existing Ranalisma rostratum, the application, which provides one kind, can be achieved asexual artificial propagation, high survival rate
Ranalisma rostratum micro-propagation method.
To achieve the above object, the technical solution that the application takes is as follows:
A kind of Ranalisma rostratum micro-propagation method, the Ranalisma rostratum after cleaning is sterilized to obtain as explant
Aseptic seedling chooses micro- numerous organ in aseptic seedling and carries out culture of rootage, obtained tissue culture transplantation of seedlings.
Further, as preferred:
Micro- numerous organ is stem section, blade or the petiole of aseptic seedling, which carries out again after carrying out bud induction
Culture of rootage.It is furthermore preferred that the stem section selects the stem section for having separate living tissue.The induced medium is cultivated by MS
Base and 6BA(6- benayl aminopurine), KT(kinetin), IBA(indolebutyric acid), ZT(zeatin) at least two compositions,
At least two additions in 6BA, KT, IBA, ZT are in MS culture medium；The addition concentration of the 6BA is 0.2-4mg/L, the KT
Addition concentration be 0.2-2mg/L, the addition concentration of the IBA is 0.4-1mg/L, and the addition concentration of the ZT is 0.2-1mg/
L。
Micro- numerous organ is the stolon of aseptic seedling, and stolon breeding of directly sprouting obtains tissue-cultured seedling.
The Aseptic seedling culture base is by MS culture medium and Ticarcillin/Clavulanate Acid and/or cephalosporin, and Ticarcillin/Clavulanate Acid and/or cephalo are mould
Element addition is in MS culture medium.It is furthermore preferred that the MS culture medium adds Ticarcillin/Clavulanate Acid and/or cephalosporin after first sterilizing again.
The root media is the MS culture medium for being not added with hormone.
The pH of the MS culture medium is 5.8-6.0, chemical component and following (the working solution concentration unit: mg/ of dosage
L): a great number of elements: NH4NO3(1650), KNO3(1900), CaCl2·2H2O(440), MgSO47H2O(370), KH2PO4
(170)；Microelement: KI(0.83), H3BO3(6.2), MnSO4·4H2O(22.3), ZnSO4·7H2O(8.6), Na2MoO4·
2H2O(0.25), CuSO4·5H2O(0.025), CoCl2·6H2O(0.025)；Molysite: FeSO4·7H2O(27.8), Na2-
EDTA·2H2O(37.3)；Organic substance: inositol (100), niacin (0.5), puridoxine hydrochloride (vitamin B6) (0.5), hydrochloric acid
Thiamine (vitamin B1) (0.1), glycine (2.0).
Agar is added in the MS culture medium, the addition concentration of agar is 5.4-5.8g/L.
Detailed description of the invention
Fig. 1 is the state diagram that Ranalisma rostratum explant is inoculated with 6 days in the application；
Fig. 2 is the aseptic seedling top view obtained when explant is cultivated 30 days in the application；
Fig. 3 is the aseptic seedling side view obtained when explant is cultivated 30 days in the application；
Fig. 4 is the aseptic seedling single plant in Fig. 3；
Fig. 5 is that the aseptic seedling fibre in Fig. 3 is crawled branch；
Fig. 6 is state diagram when Ranalisma rostratum blade is cultivated 20 days in the application；
Fig. 7 is state diagram when Ranalisma rostratum petiole is cultivated 20 days in the application；
Fig. 8 is the state diagram (culture medium: MS when stem section of the Ranalisma rostratum with separate living tissue induces 30 days in the application
2.0 mg/L+KT of+6BA, 2.0 mg/L+IBA, 0.4 mg/L)；
Fig. 9 is the single plant state diagram (arrow instruction adventitious bud) that Fiber differentiation obtains in Fig. 8；
Figure 10 is take root state diagram (i.e. the bottom view of Fig. 8)；
Figure 11 is transplanting effect picture.
Specific embodiment
1. materials and methods
1.1 experimental material
Ranalisma rostratum seedling is provided by Zhejiang Forest academy of sciences stone from extensive scholar, the training of this laboratory phjytotron
It supports, experimental material uses blade, petiole and the stem section with separate living tissue of the plant.
1.2 culture mediums and condition of culture
1.2.1 MS culture medium prescription
For MS culture medium as minimal medium, chemical component and dosage are following (working solution concentration unit: mg/L): a large amount of
Element: NH4NO3(1650), KNO3(1900), CaCl2·2H2O(440), MgSO47H2O(370), KH2PO4(170)；It is micro
Element: KI(0.83), H3BO3(6.2), MnSO4·4H2O(22.3), ZnSO4·7H2O(8.6), Na2MoO4·2H2O(0.25),
CuSO4·5H2O(0.025), CoCl2·6H2O(0.025)；Molysite: FeSO4·7H2O(27.8), Na2-EDTA·2H2O
(37.3)；Organic substance: inositol (100), niacin (0.5), puridoxine hydrochloride (vitamin B6) (0.5), thiamine hydrochloride (dimension life
Plain B1) (0.1), glycine (2.0).
1.2.2 Antibiotic medium/Aseptic seedling culture base
Three kinds be can be selected as comparing cases: (1) MS+ Ticarcillin/Clavulanate Acid 200mg/L(unit is similarly hereinafter)+cephalosporin 200；(2)
MS+ Ticarcillin/Clavulanate Acid 200；(3) MS+ cephalosporin 400.
1.2.3 bud inducement cultivation base
Ten kinds be can be selected as comparison case: (4) MS+6BA 2.0mg/L(unit is similarly hereinafter)+KT 2.0+IBA 0.4；(5)
MS+6BA 4.0+KT 2.0+IBA 0.4；(6) MS+KT 4.0+IBA 0.4；(7) MS+ZT 0.2+6BA 2.0+KT 2.0+
IBA 0.4；(8) MS+ZT 0.4+IBA 0.4；(9) MS+ZT 1.0+IBA 0.4；(10) MS+ZT 0.2+KT 2.0+IBA
0.4；(11) MS+ZT 0.2+6BA 2.0+IBA 0.4；(12) MS+ZT 0.2+IBA 1.0；(13) MS+6BA 0.2+KT 0.2
+IBA 1.0。
1.2.4 root media: not plus the MS culture medium of hormone.
Sucrose 30g/L, pH value 5.8-6.0 are all added in above-mentioned all MS culture mediums；Since Ranalisma rostratum is marsh
Plant, we add the adjustment that concentration carries out culture medium hardness by control agar, and the present embodiment is with the agar of point three class
For concentration, it is respectively: the soft culture medium of 5.4 g/L, intensity: 1000；The normal incubation medium of 5.6g/L；The hard training of 5.8 g/L
Support base；Medium sterilization condition: 115 DEG C, 15min, culture medium has gone out after bacterium, is cooled to 60 DEG C of additions Ticarcillin/Clavulanate Acid, cephalosporins etc.
Antibiotic.
1.2.5 condition of culture
Temperature in tissue culture room and artificial climate room is 25 ± 2 DEG C；Periodicity of illumination is that 14 hours illumination/10 hour are black
Secretly, intensity of illumination is 2000-2500 lux.
Induction of 1 different hormone combinations of table to stem section adventitious bud
| Number | Bud number/explant | Bud induction rate (100%) | Significant difference | Seedling state |
| (4) | 2.40 | 95 | ** | Plant is big, and blade is round, turns to be yellow a little, no stolon. |
| (5) | 2.00 | 85 | * | Plant is larger, and blade is long and narrow, turns to be yellow a little, there is stolon. |
| (6) | 1.90 | 95 | ** | Plant is larger, and blade is oval, emerald green, no stolon. |
| (7) | 2.15 | 95 | ** | Plant is smaller, and blade is long and narrow, emerald green, there is stolon. |
| (8) | 1.65 | 85 | ** | Plant is larger, and blade is round, and blade tip jaundice has stolon. |
| (9) | 1.50 | 80 | >0.05 | Plant is small, and blade is long and narrow, and peak green has stolon. |
| (10) | 0.85 | 70 | >0.05 | Plant is smaller, and blade is round, peak green, no stolon. |
| (11) | 0.95 | 70 | >0.05 | Plant is smaller, and blade is long and narrow, turns to be yellow a little, there is stolon. |
| (12) | 1.95 | 90 | ** | Plant is big, and blade is long and narrow, has a yellowing, there is stolon. |
| (13) | 1.85 | 90 | ** | Plant is larger, and blade ellipse, peak green has stolon. |
Number in table 1 refers to that the number of culture medium, each parameter in table 1 are to count for inoculation 20 days, each combination system
21 pieces of explants are counted, diversity factor are detected using t-test method, setting each explant has 1 adventitious bud as reference；" * " is represented
P < 0.05, " * * " represent p < 0.01.
1.3 method
Micro- numerous process of the application is specific as follows:
1.3.1 the culture of explant sterilizing and aseptic seedling: Ranalisma rostratum removes mud, old root and leaf, with originally
Water rinses two hours, and dense dish washing liquid solution impregnates 5 minutes, constantly shakes, and clear water rinses out dish washing liquid and moves back into superclean bench.
Explant with mercuric chloride (0.1%) sterilize 8 minutes, sterile water wash 5 minutes (being repeated four times), aseptic water washing three times, cleared liter
Mercury.Then the stem section with tender leaf is moved into the culture medium of the different antibiotic of 3 kinds of additions, judges the effect of degerming after a week.
Then the culture medium more renewed every 2 weeks, the MS that antibiotic-free is moved into after 3 times, which is cultivated, to be concentrated, and judges the effect of degerming after a week.
1.3.2 bud inducement cultivation: in superclean bench, the leaf of aseptic seedling is cut into the leaf dish of 0.5cm x 0.5cm length,
Petiole is cut into the petiole section of 0.5cm length, the stem section of the longitudinal sectional 0.2cm-0.4cm diameter of the stem with separate living tissue, these stems
Section moves into bud inducement cultivation base, cultivates in tissue culture room.The case where statistics bud induction in 20-30 days.
1.3.3 culture of rootage: when bud grows to 1.5-3cm, bud is scaled off move into it is raw plus in the MS culture medium of hormone
Root.
1.3.4 tissue culture transplantation of seedlings: when tissue-cultured seedling adventitious root grows to about 0.5 cm, moving into greenhouse, open lid, is added suitable
Measure tap water, separate the mushroom in air, dispose the culture medium of root after 3 days, plant in culture substrate (vermiculite: peat=
In 1:1), 20 days statistics survival rates.
1.4 statistical method
Data statistics is carried out using 22.0 software of SPSS, significant difference uses Student's t test." * " is indicated
Significant difference (p < 0.05)；" * * " indicates that difference is extremely significant (p < 0.01).
2. result
The acquisition of 2.1 sterile tissue-cultured seedling
We remove the intracorporal endophyte of Ranalisma rostratum, both discoveries mixing using Ticarcillin/Clavulanate Acid and cephalosporin
(i.e. culture medium (1) effect in 1.2.2 is best (referring to Fig. 1), subculture 3 times in antibiotic, then in sterile culture medium for use
Upper culture counts after 2 weeks, and explant degerming efficiency reaches 85%(153 aseptic seedling/180 explant), aseptic seedling is shown in Fig. 2-
5.And other 2 culture mediums all grow bacterial plaque generally in Initial culture, Aseptic seedling culture effect does not have culture medium (1)
It is good.And the agar of various concentration is larger to the growth effect of aseptic seedling, most preferably agar concentration is 5.4g/L's to growth conditions
Soft culture medium.In this culture medium, Aseptic Seedling Growth is in good condition, and discovery has the generation of stolon at 30 days, and grows
New adventitious bud (Fig. 5).
The induction of 2.2 adventitious buds
Generation of the blade, petiole of Ranalisma rostratum in 10 culture mediums of all designs all without callus and organ
(Fig. 6, Fig. 7), the only stem section with separate living tissue can induce out adventitious bud, the also not generation (Fig. 9, Figure 10) of callus.It is logical
Cross culture medium (4) bud inducing amount in the quantity discovery 1.2.3 to culture medium (table 1) evoking adventive bud of hormon formula
At most, average each explant reaches 2.4 adventitious buds (Fig. 8).With rush cell division hormone (ZT, 6BA and KT) total amount
Increase, inhibit the induction (culture medium (5), (6) and (7) in table 1) of adventitious bud, when hormone-content is low in culture medium, it is indefinite to induce
The efficiency of bud (culture medium (8), (9) and (10) in table 1) is lower, when IBA increases in culture medium, then can promote the generation of adventitious bud
(culture medium (11) in table 1, (12) and (13)).
2.3 taking root
Tissue culture during adventitious bud has different degrees of phenomenon of taking root, especially aseptic seedling to obtain in different medium
Seedling rooting is all right, we are transferred to the adventitious bud turned out in root media (MS) and cultivate, rooting rate up to 95% with
Upper (Figure 10).
2.4 tissue culture transplantation of seedlings
Ranalisma rostratum is planted into matrix, is covered transparent cover moisturizing, is put into phjytotron culture.After 2 days, plant
Object blade starts upright and returns green, takes lid away, opens fluorescent lamp, allows its normal growth.Later period plant strain growth accelerates, and blade becomes
Green, growing way is vigorous.Statistics transplanting survival rate is in 95% or more (Figure 11) within 20 days.
There are two types of the modess of reproduction of Ranalisma rostratum: asexual and sexual, the application is when obtaining sterile tissue-cultured seedling, discovery
Have the generation of stolon, and have adventitious root generation on stolon, eventually form succession of rectilinear arrangement new plant (Fig. 3 and
Fig. 5).This illustrates the mode of nourishing and generating naturally that can simulate Ranalisma rostratum, by tissue culture culture in glassware stolon come micro-
Numerous Ranalisma rostratum.
The concentration of plant hormone and, the differentiation of basic element of cell division promotion bud very big with comparison Plant Tissue Breeding influence, life
Long element promotes calli induction, its suitable concentration proportioning of different plants is different.We have found that with separate living tissue
Stem can induce out adventitious bud, and blade and petiole induce callus and the effect of adventitious bud (Fig. 6 and Fig. 7) is unobvious.Therefore,
It can be used as one of the mode of Ranalisma rostratum artificial propagation by micro- numerous mode of stem section evoking adventive bud.
The application by the research to aseptic seedling, culture medium hardness, adventitious bud inducing, the process conditions such as take root and transplant,
In above-mentioned each cultural method, the induction of the micro- numerous mode of stolon and the micro- numerous two kinds of micro-propagation methods of mode of stem section adventitious bud in tissue culture bottle
Effect is best, most effective, can provide effective technology for the protection of the species.
The above content is the preferred embodiments of combination the invention to further detailed made by provided technical solution
Describe in detail bright, and it cannot be said that the invention specific implementation is confined to these above-mentioned explanations, technology affiliated for the invention
For the those of ordinary skill in field, without departing from the concept of the premise of the invention, several simple deductions can also be made
Or replacement, it all shall be regarded as belonging to the protection scope of the invention.
Claims (5)
1. a kind of Ranalisma rostratum micro-propagation method, it is characterised in that: using the Ranalisma rostratum after cleaning as explant into
Row sterilizing culture obtains aseptic seedling, and micro- numerous organ is chosen in aseptic seedling and carries out bud inducement cultivation and culture of rootage, obtained group
Train transplantation of seedlings；The bud inducement cultivation be with bud inducement cultivation base culture, the bud inducement cultivation base by MS culture medium,
6BA, KT, IBA, agar and sucrose composition；The addition concentration of the 6BA is 0.2-4mg/L, and the addition concentration of the KT is 0.2-
The addition concentration of 2mg/L, the IBA are 0.4-1mg/L；The culture of rootage is with root media culture, and described takes root
Culture medium is the MS culture medium for being not added with hormone.
2. a kind of Ranalisma rostratum micro-propagation method as described in claim 1, it is characterised in that: micro- numerous organ is nothing
The stolon of vaccine, stolon breeding of directly sprouting obtain tissue-cultured seedling.
3. a kind of Ranalisma rostratum micro-propagation method as described in claim 1, it is characterised in that: the sterilizing culture is with nothing
Vaccine culture medium culture, the Aseptic seedling culture base are made of MS culture medium and Ticarcillin/Clavulanate Acid and/or cephalosporin, the special beauty
Spit of fland and/or cephalosporin addition are in MS culture medium.
4. a kind of Ranalisma rostratum micro-propagation method as described in claim 1, it is characterised in that: the pH of the MS culture medium
For 5.8-6.0, chemical component and dosage are as follows: NH4NO3: 1650 mg/L, KNO3: 1900 mg/L, CaCl2·2H2O:440
Mg/L, MgSO4·7 H2O:370 mg/L, KH2PO4: 170 mg/L；KI:0.83 mg/L, H3BO3: 6.2 mg/L, MnSO4·
4H2O:22.3 mg/L, ZnSO4·7H2O:8.6 mg/L, Na2MoO4·2H2O:0.25 mg/L, CuSO4·5H2O:0.025
Mg/L, CoCl2·6H2O:0.025 mg/L；FeSO4·7H2O:27.8 mg/L, Na2-EDTA·2H2O:37.3 mg/L；Flesh
Alcohol: 100 mg/L, niacin: 0.5 mg/L, puridoxine hydrochloride: 0.5 mg/L, thiamine hydrochloride: 0.1 mg/L, glycine: 2.0
mg/L。
5. a kind of Ranalisma rostratum micro-propagation method as described in claim 1, it is characterised in that: the addition concentration of the agar
For 5.4-5.8g/L.
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| CN104206280A (en) * | 2014-09-21 | 2014-12-17 | 云南省农业科学院花卉研究所 | Tissue culture and rapid propagation method of hemionitis arifolia seedlings by virtue of green spherical body manner |
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| CN104206280A (en) * | 2014-09-21 | 2014-12-17 | 云南省农业科学院花卉研究所 | Tissue culture and rapid propagation method of hemionitis arifolia seedlings by virtue of green spherical body manner |
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